马氏珠母贝(Pinctada fucata martensii)Pm-HK基因的克隆及其对温度胁迫的响应

己糖激酶(hexokinase, HK)是糖酵解过程中的首个限速酶。本实验利用RACE技术克隆获得了马氏珠母贝HK (Pm-HK)基因,并对其基因和氨基酸序列结构特征进行了生物信息学分析,同时采用荧光定量PCR技术分析了该基因在不同组织的表达模式及不同温度下的时序表达模式。序列分析表明,Pm-HK cDNA序列全长为2 403 bp,其中开放式阅读框1 548 bp,5'UTR 74 bp,3'UTR 781 bp,共编码515个氨基酸,分子量为57.67 kD,理论等电点为6.08。结构域分析发现Pm-HK具有两个完整的保守功能域Hexokinase_1、Hexokinase_2,以及两个Glucose-6-Phosphate结合位点、三个Glucose结合位点,两个ATP结合位点。多序列比对结果表明Pm-HK与太平洋牡蛎HK同源性最高,为81%;系统进化分析发现,Pm-HK与太平洋牡蛎等贝类HK聚为一支。组织表达定量分析结果显示,Pm-HK在马氏珠母贝肝胰腺中显著高表达;对不同温度下鳃组织中Pm-HK的时序表达分析发现,在处理12 h后各时间点低温组(12℃) Pm-HK基因表达水平最高,且均显著高于中低温组(17℃)、对照组(22℃)和高温组(32℃)。以上结果表明Pm-HK可能参与马氏珠母贝的低温胁迫响应。该研究为进一步探索Pm-HK在马氏珠母贝在温度适应性中的作用提供了基础资料。

Cloning of Pm-HK Gene from Pinctada fucata martensii and Its Response to Temperature Stress

Hexokinase(HK) is the first rate-limiting enzyme in the glycolysis process. In this study,%hexokinase of Pinctada fucata martensii(Pm-HK) was cloned by RACE technology, the structural characteristics of the gene and amino acid sequence were analyzed by bioinformatics, and the expression patterns of Pm-HK in different tissues as well as its temporal expression patterns under different temperatures were analyzed by fluorescence quantitative PCR technology. Sequence analysis showed that the full length of Pm-HK cDNA was 2 403 bp, containing 1 548 bp of the ORF, 74 bp of 5’UTR and 781 bp of 3’UTR, which encoded 515 amino acids. The molecular weight was57.67 kD, and the theoretical isoelectric point was 6.08. Domain analysis revealed that Pm-HK had two intact conserved domains, Hexokinase1 and Hexokinase2, two Glucose-6-Phosphate binding sites, three Glucose bindingsites, and two ATP binding sites. Multiple sequence alignment results showed that Pm-HK had the highest homology with Hexokinase(HK) in Crassostrea gigas, which was 81%. Phylogenetic analysis found that Pm-HK was clustered with shellfish HK such as Crassostrea gigas. The results of the quantitative analysis of tissue expression showed that Pm-HK was significantly overexpressed in hepatopancreas of Pinctada fucata martensii. The analysis of Pm-HK temporal expression in gill tissues under different temperatures indicated that Pm-HK gene showed highest expression level in low temperature group(12℃) 12 h after treatment, which was significantly higher than that in medium and low temperature group(17℃), control group(22℃) and high temperature group(32℃). The results suggested that Pm-HK might be involved in the resistance to low-temperature stress of Pinctada fucata martensii. This study might provide basic data for the further study on the role of Pm-HK in temperature adaptability of Pinctada fucata martensii.